Erratum: Constellation: a tool for rapid, automated phenotype assignment of a highly polymorphic pharmacogene, CYP2D6, from whole-genome sequences.

[This corrects the article DOI: 10.1038/npjgenmed.2015.7.].

In vivo characterization of CYP2D6*12, *29 and *84 using dextromethorphan as a probe drug: a case report.

CYP2D6*84 was first described in a Black South African subject, however, its function remains unknown. Astrolabe, a probabilistic scoring tool developed in our laboratory to call genotypes from whole genome sequence, identified CYP2D6*84 in a trio. The father presented with intermediate metabolism when challenged with the CYP2D6 probe drug dextromethorphan (DM/dextrorphan [DX] = 0.0839). Since his second allele, CYP2D6*12, is nonfunctional, the observed activity is derived by CYP2D6*84. This finding suggests that the allele's hallmark P267H causes decreased activity toward DM and that this allele should receive a value of 0.5 for Activity Score calculations. The mother's DM/DX of 0.0543 was consistent with the decreased activity classification of CYP2D6*29. The child, a critically ill neonate, was not phenotyped, but predicted to be a normal metabolizer.

Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation.

Although CYP2B6 catalyzes the biotransformation of many drugs used clinically for children and adults, information regarding the effects of development on CYP2B6 expression and activity are scarce. Utilizing a large panel of human liver samples (201 donors: 24 fetal, 141 pediatric, and 36 adult), we quantified CYP2B6 mRNA and protein expression levels, characterized CYP2B6 (bupropion hydroxylase) activity in human liver microsomes (HLMs), and performed an extensive genotype analysis to differentiate CYP2B6 haplotypes such that the impact of genetic variation on these parameters could be assessed. Fetal livers contained extremely low levels of CYP2B6 mRNA relative to postnatal samples and fetal HLMs did not appear to catalyze bupropion hydroxylation; however, fetal CYP2B6 protein levels were not significantly different from postnatal levels. Considerable interindividual variation in CYP2B6 mRNA expression, protein levels, and activity was observed in postnatal HLMs (mRNA, ∼40,000-fold; protein, ∼300-fold; activity, ∼600-fold). The extremely wide range of interindividual variability in CYP2B6 expression and activity was significantly associated with age (P < 0.01) following log transformation of the data. Our data suggest that CYP2B6 activity appears as early as the first day of life, increases through infancy, and by 1 year of age, CYP2B6 levels and activity may approach those of adults. Surprisingly, CYP2B6 interindividual variability was not significantly associated with genetic variation in CYP2B6, nor was it associated with differences in gender or ethnicity, suggesting that factors other than these are largely responsible for the wide range of variability in CYP2B6 expression and activity observed among a large group of individuals/samples.

Constellation: a tool for rapid, automated phenotype assignment of a highly polymorphic pharmacogene, CYP2D6, from whole-genome sequences.

An important component of precision medicine-the use of whole-genome sequencing (WGS) to guide lifelong healthcare-is electronic decision support to inform drug choice and dosing. To achieve this, automated identification of genetic variation in genes involved in drug absorption, distribution, metabolism, excretion and response (ADMER) is required. CYP2D6 is a major enzyme for drug bioactivation and elimination. CYP2D6 activity is predominantly governed by genetic variation; however, it is technically arduous to haplotype. Not only is the nucleotide sequence of CYP2D6 highly polymorphic, but the locus also features diverse structural variations, including gene deletion, duplication, multiplication events and rearrangements with the nonfunctional, neighbouring CYP2D7 and CYP2D8 genes. We developed Constellation, a probabilistic scoring system, enabling automated ascertainment of CYP2D6 activity scores from 2×100 paired-end WGS. The consensus reference method included TaqMan genotyping assays, quantitative copy-number variation determination and Sanger sequencing. When compared with the consensus reference Constellation had an analytic sensitivity of 97% (59 of 61 diplotypes) and analytic specificity of 95% (116 of 122 haplotypes). All extreme phenotypes, i.e., poor and ultrarapid metabolisers were accurately identified by Constellation. Constellation is anticipated to be extensible to functional variation in all ADMER genes, and to be performed at marginal incremental financial and computational costs in the setting of diagnostic WGS.

A 26-hour system of highly sensitive whole genome sequencing for emergency management of genetic diseases.

While the cost of whole genome sequencing (WGS) is approaching the realm of routine medical tests, it remains too tardy to help guide the management of many acute medical conditions. Rapid WGS is imperative in light of growing evidence of its utility in acute care, such as in diagnosis of genetic diseases in very ill infants, and genotype-guided choice of chemotherapy at cancer relapse. In such situations, delayed, empiric, or phenotype-based clinical decisions may meet with substantial morbidity or mortality. We previously described a rapid WGS method, STATseq, with a sensitivity of >96 % for nucleotide variants that allowed a provisional diagnosis of a genetic disease in 50 h. Here improvements in sequencing run time, read alignment, and variant calling are described that enable 26-h time to provisional molecular diagnosis with >99.5 % sensitivity and specificity of genotypes. STATseq appears to be an appropriate strategy for acutely ill patients with potentially actionable genetic diseases.

Whole-genome sequencing for identification of Mendelian disorders in critically ill infants: a retrospective analysis of diagnostic and clinical findings.

BACKGROUND: Genetic disorders and congenital anomalies are the leading causes of infant mortality. Diagnosis of most genetic diseases in neonatal and paediatric intensive care units (NICU and PICU) is not sufficiently timely to guide acute clinical management. We used rapid whole-genome sequencing (STATseq) in a level 4 NICU and PICU to assess the rate and types of molecular diagnoses, and the prevalence, types, and effect of diagnoses that are likely to change medical management in critically ill infants. METHODS: We did a retrospective comparison of STATseq and standard genetic testing in a case series from the NICU and PICU of a large children's hospital between Nov 11, 2011, and Oct 1, 2014. The participants were families with an infant younger than 4 months with an acute illness of suspected genetic cause. The intervention was STATseq of trios (both parents and their affected infant). The main measures were the diagnostic rate, time to diagnosis, and rate of change in management after standard genetic testing and STATseq. FINDINGS: 20 (57%) of 35 infants were diagnosed with a genetic disease by use of STATseq and three (9%) of 32 by use of standard genetic testing (p=0·0002). Median time to genome analysis was 5 days (range 3-153) and median time to STATseq report was 23 days (5-912). 13 (65%) of 20 STATseq diagnoses were associated with de-novo mutations. Acute clinical usefulness was noted in 13 (65%) of 20 infants with a STATseq diagnosis, four (20%) had diagnoses with strongly favourable effects on management, and six (30%) were started on palliative care. 120-day mortality was 57% (12 of 21) in infants with a genetic diagnosis. INTERPRETATION: In selected acutely ill infants, STATseq had a high rate of diagnosis of genetic disorders. Most diagnoses altered the management of infants in the NICU or PICU. The very high infant mortality rate indicates a substantial need for rapid genomic diagnoses to be allied with a novel framework for precision medicine for infants in NICU and PICU who are diagnosed with genetic diseases to improve outcomes. FUNDING: Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Human Genome Research Institute, and National Center for Advancing Translational Sciences.

A patient with polymerase E1 deficiency (POLE1): clinical features and overlap with DNA breakage/instability syndromes.

BACKGROUND: Chromosome instability syndromes are a group of inherited conditions associated with chromosomal instability and breakage, often leading to immunodeficiency, growth retardation and increased risk of malignancy. CASE PRESENTATION: We performed exome sequencing on a girl with a suspected chromosome instability syndrome that manifested as growth retardation, microcephaly, developmental delay, dysmorphic features, poikiloderma, immune deficiency with pancytopenia, and myelodysplasia. She was homozygous for a previously reported splice variant, c.4444 + 3A > G in the POLE1 gene, which encodes the catalytic subunit of DNA polymerase E. CONCLUSION: This is the second family with POLE1-deficency, with the affected individual demonstrating a more severe phenotype than previously described.

Effectiveness of exome and genome sequencing guided by acuity of illness for diagnosis of neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) affect more than 3% of children and are attributable to single-gene mutations at more than 1000 loci. Traditional methods yield molecular diagnoses in less than one-half of children with NDD. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) can enable diagnosis of NDD, but their clinical and cost-effectiveness are unknown. One hundred families with 119 children affected by NDD received diagnostic WGS and/or WES of parent-child trios, wherein the sequencing approach was guided by acuity of illness. Forty-five percent received molecular diagnoses. An accelerated sequencing modality, rapid WGS, yielded diagnoses in 73% of families with acutely ill children (11 of 15). Forty percent of families with children with nonacute NDD, followed in ambulatory care clinics (34 of 85), received diagnoses: 33 by WES and 1 by staged WES then WGS. The cost of prior negative tests in the nonacute patients was $19,100 per family, suggesting sequencing to be cost-effective at up to $7640 per family. A change in clinical care or impression of the pathophysiology was reported in 49% of newly diagnosed families. If WES or WGS had been performed at symptom onset, genomic diagnoses may have been made 77 months earlier than occurred in this study. It is suggested that initial diagnostic evaluation of children with NDD should include trio WGS or WES, with extension of accelerated sequencing modalities to high-acuity patients.

High-resolution melt analysis to detect sequence variations in highly homologous gene regions: application to CYP2B6.

High-resolution melt (HRM) analysis using 'release-on-demand' dyes, such as EvaGreen(®) has the potential to resolve complex genotypes in situations where genotype interpretation is complicated by the presence of pseudogenes or allelic variants in close proximity to the locus of interest. We explored the utility of HRM to genotype a SNP (785A>G, K262R, rs2279343) that is located within exon 5 of the CYP2B6 gene, which contributes to the metabolism of a number of clinically used drugs. Testing of 785A>G is challenging, but crucial for accurate genotype determination. This SNP is part of multiple known CYP2B6 haplotypes and located in a region that is identical to CYP2B7, a nonfunctional pseudogene. Because small CYP2B6-specific PCR amplicons bracketing 785A>G cannot be generated, we simultaneously amplified both genes. A panel of 235 liver tissue DNAs and five Coriell samples were assessed. Eight CYP2B6/CYP2B7 diplotype combinations were found and a novel variant 769G>A (D257N) was discovered. The frequency of 785G corresponded to those reported for Caucasians and African-Americans. Assay performance was confirmed by CYP2B6 and/or CYP2B7 sequence analysis in a subset of samples, using a preamplified CYP2B6-specific long-range-PCR amplicon as HRM template. Inclusion rather than exclusion of a homologous pseudogene allowed us to devise a sensitive, reliable and affordable assay to test this CYP2B6 SNP. This assay design may be utilized to overcome the challenges and limitations of other methods. Owing to the flexibility of HRM, this assay design can easily be adapted to other gene loci of interest.

CYP2D6, SULT1A1 and UGT2B17 copy number variation: quantitative detection by multiplex PCR.

AIM: Among the genes of drug-metabolizing enzymes, CYP2D6 is notoriously difficult to characterize owing to the complexity of gene deletions, duplications, multiplications and the presence of hybrid genes composed of CYP2D6 and CYP2D7. For SULT1A1 up to five gene copies have been reported, while UGT2B17 is known for gene deletions only. Different platforms exist for copy number variation (CNV) detection; however, there are no gold standards. Robust methods are required that address specific challenges to accurately determine gene CNVs in complex gene loci. MATERIALS & METHODS: Quantitative multiplex PCR amplification (MPA) was performed on a diverse set of genomic DNA samples. Resulting PCR fragments were separated on an ABI 3730 instrument and analyzed with GeneMapper. CYP2D6 was targeted at four different gene regions and either normalized against CYP2D8 or UGT2B15 and SULT1A2. Inconsistent observations and CNVs contrasting genotype data were further characterized by long-range PCR and/or DNA sequence analysis. UGT2B17 and SULT1A1 were normalized against UGT2B15 and SULT1A2, respectively. RESULTS: MPA detected 0-5, 1-5 and 0-2 copies for CYP2D6, SULT1A1 and UGT2B17, respectively. The interrogation of four CYP2D6 regions resulted in robust copy number assignments that were in agreement with genotype, sequencing and extra long PCR-based data. Gene deletions, duplication, and multiplications among known and novel hybrid genes were reliably identified. Novel findings regarding allelic variation include nonfunctional CYP2D6/2D7 hybrids such as CYP2D6*4N and *68, which were consistently identified on a subset of CYP2D6*4 alleles. In addition, a novel variant, designated CYP2D6*83, was discovered. For SULT1A1, we report the first six-copy case and for UGT2B15 and UGT2B17 we have evidence for rare deletion and duplication events, respectively. CONCLUSION: This MPA-based copy number platform not only allowed us to determine CNVs, but also served as a tool for allele discovery and characterization in a diverse panel of samples in a fast and reliable manner.